Method of activating immune response in plants

ABSTRACT

A method of enhancing plant immunity is provided. The method comprises the step of administering to a plant a small molecule that binds to NPR1, or a functionally equivalent homolog thereof, that disrupts the interaction between N-terminal BTB/POZ domain and the C-terminal transactivation domain of NPR1. A method of screening for small molecule compounds that enhance plant immunity is also provided.

CROSS REFERENCE TO RELATED APPLICATIONS

This application claims the benefit of U.S. Provisional PatentApplication No. 61/657,461, filed Jun. 8, 2012, the disclosure of whichis incorporated fully herein by reference.

FIELD OF THE INVENTION

The present invention relates to Systemic Acquired Resistance (SAR) inplants, and more particularly, to methods of activating plant immuneresponse.

BACKGROUND OF THE INVENTION

Salicylic acid (SA) serves as an endogenous phytohormone in thedeployment of Systemic Acquired Resistance (SAR), a broad-spectrum andlong-lasting immune response activated by avirulent pathogens in plants.Its deployment is monitored through the marker gene PR-1, whoseactivation requires the recruitment of an SA-dependent transcriptionalenhanceosome to its promoter. The enhanceosome contains members of theTGA2 Glade of bZIP transcription factors and the transcriptionalcoactivator NPR1, which is the central regulator of SAR and SA-dependentgene activation. TGA2 is a transcriptional repressor and thus requires acoactivator to effect gene activation. NPR1 provides a dual function inthe enhanceosome. First, its N-terminal region contains a BTB/POZ domainwhich interacts with and negates the function of the TGA2 repressiondomain, and secondly, NPR1 harbors in its C-terminal region atransactivation domain, which contains two cysteines (Cys⁵²¹ and Cys⁵²⁹)required for the activating function of the enhanceosome.

In non-SA-stimulated cells, NPR1-GFP fusion proteins behave as oligomerson sodium dodecyl-sulfate-polyacrylamide gel (SDS-PAGE) electrophoresis.Endogenous NPR1 localizes to both the nucleus and the cytosol andnuclear localization is critical to PR-1 activation. A fraction of thenuclear NPR1 population acts as a latent coactivator which is recruitedunder non-inducing conditions to the PR-1 promoter. There thus exists anuncharacterized mechanism by which the NPR1 transactivating domainremains occluded under non-inducing conditions and gets unveiled duringSA-dependent gene activation. Furthermore, although genetic analyseshave revealed many genes involved in SA-signaling, the receptorresponsible for sensing SA and leading to direct or indirect NPR1activation remains elusive.

While enzymes, such as catalase, peroxidase, and methyl-salicylateesterase, have been shown to directly interact with SA, their proposedrole in SAR has not been unequivocal. SA was originally portrayed as acatalase and peroxidase inhibitor, leading to the generation of H₂O₂,and the production of PR protein. However, H₂O₂ was later shown not tobe a second messenger acting downstream of SA, invalidating the role ofcatalase and peroxidase as SA-receptors for PR gene activation. Whereasmethyl-salicylate esterase has been shown to play a role in tobacco, itclearly has no role in SAR in Arabidopsis. Most importantly, theseenzymes do not figure as classical transcription regulators andtherefore, they are unlikely to regulate gene expression directly.

It would be desirable to develop novel methods of activating the immuneresponse in plants in order to enhance immunity in plants.

SUMMARY OF THE INVENTION

It has now been determined that NPR1 is the receptor for salicylic acid,and specifically interacts with salicylic acid. This determinationpermits the targeted enhancement of plant immunity, as well as theidentification of potential plant immunity-enhancing compounds.

Thus, in one aspect of the present invention, a method of enhancingplant immunity is provided comprising the step of administering to aplant a small molecule that binds to NPR1, or a functionally equivalentsalicylic acid-binding protein, and disrupts the interaction betweenN-terminal BTB/POZ domain and the C-terminal transactivation domain ofthe NPR1 protein.

In another aspect, a method of identifying small molecule compounds thatenhance plant immunity is provided. The method comprises the step ofscreening a candidate compound for binding to the NPR1 C-terminaltransactivation domain and determining whether or not the compound bindsto the NPR1 protein, wherein a compound that exhibits a binding affinityfor the NPR1 C-terminal transactivation domain is a candidate compoundthat may enhance plant immunity.

These and other aspects of the invention are described in the detaileddescription by reference to the following figures.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1. [¹⁴C]SA binding of no protein, NPR1, the BTB/POZ of NPR1 (POZ),or the transactivation domain of NPR1 (Δ513) using (A) a classicalnon-equilibrium solid-phase method or (B) equilibrium dialysis; (C)saturation binding of [¹⁴C]SA to NPR1 using equilibrium dialysis; (D)Scatchard Plot of the data in (C); (E) Homologous and (F) heterologouscompetitive binding curves for the [¹⁴C]SA-NPR1 interaction usingequilibrium dialysis; (G) illustrates structures of the competitors; (H)Quantitative RT-PCR; and (I) Fold-induction of a given chemicaltreatment over a water control, calculated using data from (H).

FIG. 2. (A) Sequence of the NPR1 transactivation domain (Δ513) showingCys⁵²¹ and Cys⁵²⁹ (note that NPR1 ends at amino acid 593); (B-E)immunoblots of HA-tagged NPR1 transactivation domains separated byimmobilized metal-affinity chromatography (Ni-NTA) in the presence ofeither buffer alone, or buffer supplemented with EDTA; (F)[¹⁴C]SA-binding assays of the transactivation domain of NPR1 (Δ513)using equilibrium dialysis; and Concentrations of various candidated-block metals associated with E. coli-produced (G) andArabidopsis-produced (H) NPR1 protein (WT/mutant).

FIG. 3. Immunoblot analysis of protein fractions from an S300 elutionprofile of (A) untreated, (B) SA-treated, (E) EDTA and SA-treated, (F)Catechol-treated, (G) 4-hydroxy-benzoic acid-treated, (H)methyl-salicylate-treated or (I) DTT-treated Strep-tagged NPR1, using ananti-Strep antibody; Immunoblot analysis, of protein fractions from anS300 elution profile of (C) untreated or (D) SA-treated Strep-taggedNPR1 harboring a Cys-to-Ser substitution; Coomassie stain of an SDS-PAGEgel showing purified NPR1 and the NPR1 mutant bearing acysteine-to-serine substitution at positions 521 and 529 (J); and 3CMethod showing the presence/absence of NPR1-dependent oligomer on thePR-1 promoter.

FIG. 4. S100 gel filtration chromatogram illustrating the elutionprofile of purified Strep-tagged Δ513 (A) untreated or (C) treated with1 mM SA; and Immunoblot, using an anti-Strep antibody, of fractions (Band D) corresponding to theoretical peaks and taken from thechromatograms in (A) and (C).

FIG. 5. (A) In vivo transcription assays showing that the NPR1C-terminal transactivation domain (Δ513:DB) can activate thetranscription of a reporter gene in the absence of SA-treatment; (B) Invivo plant two-hybrid assays showing that Δ513:DB can only interact withthe NPR1 BTB/POZ domain (POZ:DB) in the absence of SA; (C) Pull-downassay using the BTB/POZ fused to the Strep-Tag and coupled to theStrepTactin solid-phase and the Δ513 fused to the HA-Tag; (D) Pull-downassay using the BTB/POZ fused to the Strep-Tag and coupled to theStrepTactin solid-phase and the VLRSgt protein (a glucosyltransferaseunrelated in sequence to Δ513) fused to the GST-tag; (E) Pull-down assayusing the empty StrepTactin solid-phase and the Δ513 fused to theHA-Tag; (F) In vivo transcription assays testing the transactivationproperties of Δ513:DB, both alone and in complex with the NPR1 BTB/POZ(POZ) not fused to any domain; (G) In vivo transcription assaysassessing the potential transcriptional repression conferred by the NPR1BTB/POZ domain (POZ) tethered to DNA through the Gal4 DNA-binding domain(:DB).

FIG. 6. Schematic illustrating the interaction between NPR1's C-TerminalTransactivation Domain (Δ513) and N-Terminal Auto-Inhibitory Domain(BTB/POZ).

FIG. 7. Schematic of the NPR1 structure (A); Saturation binding of[¹⁴C]SA to untagged NPR1 using equilibrium dialysis (B); Scatchard Plot(C) of the data in (B); Saturation binding of [¹⁴C]SA to NPR1 usingScintillation Proximity Assay (D); Scatchard Plot (E) of the data in(D); Homologous and heterologous competitive binding curves for the[¹⁴C]SA-NPR1 interaction using Scintillation Proximity Assay (F/G).

FIG. 8. Schematics and outcomes of the BTB/POZ-Δ513 pull-down assays.The boxed diagram indicates the outcome observed in FIG. 5C.

FIG. 9. Amino acid sequences of the C-terminus of NPR1 protein fromdifferent plants.

DETAILED DESCRIPTION OF THE INVENTION

The present invention relates to a method of enhancing plant immunity byPR-1 gene activation. The method comprises the step of administering toa plant a small molecule that binds to NPR1, or binds to a functionallyequivalent salicylic acid receptor protein (NPR), and disturbs theinteraction between N-terminal BTB/POZ domain and the C-terminaltransactivation domain of NPR1.

The term “NPR1” or “NIM1” refers to a plant transcriptional coactivatorthat is involved in PR (pathogenesis-related) gene activation. The term“NPR1” as used herein is meant to encompass NPR1 proteins in plantsincluding, but not limited to, Arabidopsis, Theobroma, tobacco, cotton,rice, legume and the like. Amino acid sequences of the C-terminaltransactivation domain of NPR1 are set out in FIG. 9, and include thesequence of the C-terminus of the NPR1 protein from Arabidopsis thaliana(AtNPR1), as well as the sequence of the C-terminus of NPR1 receptorproteins in Theobroma cacao (TcNPR1), Gossypium hirstum(GhNPR1)(cotton), Nicotiana tabacum (NtNPR1)(tobacco), and Oryza sativa(OsNPR1)(rice), as well as functionally equivalent NPR receptors. Theterm “functionally equivalent” as it is used herein is meant to refer toother NPR salicylic acid receptor proteins in plants, such as NPR5 andNPR6.

The present method includes the step of PR gene activation byadministration of a small molecule that binds to a salicyclic acidreceptor protein, such as NPR1, and disturbs the interaction betweenN-terminal BTB/POZ domain and the C-terminal transactivation domain ofNPR1, or a functionally equivalent salicylic acid receptor protein. Theterm “small molecule” refers to a molecule having a molecular weight ofless than 5 kilodaltons (kD), preferably less than 2.5 kD, and morepreferably, a molecule having a molecular weight of 1 kD or less, andare herein referred to as “NPR-binding”.

An NPR-binding small molecule in accordance with the present inventionmay have the following general formula:

wherein X and Y are each an electronegative functional group thattogether can coordinate a transition metal such as copper; and ring A isa hydrophobic cyclic core. Examples of electronegative functional groupsinclude groups containing, for example, oxygen, nitrogen and sulfur,such as hydroxyl, carbonyl, amine, —NHR wherein R is a lower alkylgroup, nitro, —SH, —COH, —OCOH and —CH₂OH.

Ring A may, for example, be selected from the group consisting ofphenyl, heterocyclyl, cyclohexyl and cyclopentyl, and may optionally besubstituted with one or more groups selected from, for example, halogen,e.g. chlorine, fluorine, bromine; hydroxyl, thio, C₁-C₆ alkyl, C₁-C₆alkyl halide, —OR¹, —NH₂, —NO₂, —NHR¹, —NR¹R² or —SR¹, fused phenyl andfused heterocyclyl. The variables R¹ and R² are independently selectedfrom the group consisting of C₁-C₆ alkyl, C₁-C₆ alkyl halide, C₁-C₆alkanol and C₁-C₆ alkoxy.

The term “heterocyclyl” is used herein to encompass ring structures thatinclude at least one hetero atom selected from O, S and N within thecore ring structure, and preferably 5- and 6-membered ring structuressuch as, but not limited to, furan, thiophene, pyrrole, pyran,pyrimidine, piperazine, thiazine and oxazine. Examples of fusedheterocyclyl-containing rings, or bicyclic hydrophobic cores include,but are not limited to, benzothiophene, quinoline, isoquinoline, indole,benzofuran and purine. An example of a fused non-heterocyclyl-containingring structure is naphthalene.

The NPR-binding small molecule is administered to the plant in anconcentration ranging from about 10-1000 micromolar, and morepreferably, a concentration from about 100 to 500 micromolar.

As one of skill in the art will appreciate, NPR-binding small moleculemay be administered to a plant in a suitable agriculturally acceptableformulation, including but not limited to, a growing medium such as soilor hydroponic liquid medium, dusts, granules, solution concentrates,emulsifiable concentrates and wettable powders. The term “agriculturallyacceptable” indicates that the formulation is non-toxic and otherwiseacceptable for application to a plant, whether applied indoors (e.g. ina contained environment) or outdoors (e.g. in a non-containedenvironment that is exposed to other plant, animal and human life). Theformulation may include additives such as solvents, for example ketones,alcohols, aliphatic ethers; surfactants, for example aliphatic alcoholsulfates, alkylphenol ethoxylates, Silwet; or other additives such asfillers and carriers, for example clay and minerals; and plant extracts,such as nut shells and guar gum.

The NPR-binding molecule may be administered to a plant in a compositionincluding one or more additional plant growth-enhancing compounds,including but not limited to, macronutrients such as a nitrogen source(e.g. ammonium, nitrates and the like), a phosphorous source (e.g.phosphoric acid), and a potassium source (e.g. potash), micronutrientssuch as boron, iron, calcium, magnesium, sulfur, selenium, manganese,molybdenum, zinc and iodine, and vitamins and cofactors such asthiamine, riboflavin, niacin (nicotinic acid and/or niacinamide),pyridoxine, panthenol, cyanocobalamin, citric acid, folic acid, biotinand combinations thereof.

While a clear advantage of the present method of enhancing plantimmunity is to minimize or avoid the use of toxic pesticides andherbicides, an NPR-binding molecule may be administered to a plant incombination with insecticides such as organophosphates, pyrethroids andneonicotinoids, inorganic materials such as aresenates, copper andsulfur, and biological control agents such as Bacillus spp.

An additional step in the method may include administration of atransition metal, e.g. copper, to the plant with or followingadministration of the selected small molecule. Soil generally containssufficient copper to coordinate with an NPR-binding small moleculeadministered to a plant; however, in the event that the soil istransition metal-free, or substantially transition metal-free (e.g. lessthan about 9 ppm transition metal content in the soil), or in the eventthat plant growing media other than soil is being used (for example,water or other liquid growing medium) that is transition metal-free,transition metal may be administered to plant in an amount of at leastabout 9 ppm, and preferably an amount in the range of 9 to 30 ppm ifapplied to the soil or other growing medium of the plant, or in amountof about 50 to 300 micromolar if applied directly to a plant in aformulation that may also include the NPR-binding molecule, or in aformulation separate from the NPR-binding molecule. In this regard,transition metal, for example copper, is generally administered to theplant or admixed with the formulation in the form of a salt, forexample, sulfate, chloride, bromide, fluoride, iodide, d-gluconate,hydroxide, molybdate, nitrate, perchlorate or thiocyanate.Alternatively, the metal may be administered as a chelate, for exampleas an ethylenediamine (EDTA), ethanolamine, triethanolamine orsalicylate chelate.

In another aspect, a method of identifying small molecule compounds thatenhance plant immunity is provided. The method comprises the step ofscreening a small molecule candidate compound for NPR binding, andspecifically, binding to the C-terminal transactivation domain of NPR1or of a functionally equivalent salicylic acid receptor protein. Thefunction of the small molecule to bind this C-terminal region of NPR1may be determined using established binding assays as described herein.For example, NPR binding may be determined using an assay in which theNPR is immobilized on a solid phase followed by treatment with adetectably labeled molecule, e.g. a radioactive, colorimetric orenzymatic label. Other methods to determine NPR1 binding may also beused, as one of skill in the art will appreciate, for example,equilibrium dialysis or isothermal titration calorimetry. Smallmolecules determined to exhibit a binding affinity to the C-terminaltransactivation domain of an NPR that is similar to that of salicylicacid for NPR1, e.g. a k_(d) of about 50 μM or less, for example, 40 μMor less, 30 μM or less, 20 μM or less, or 10 μM or less, are candidatecompounds that may enhance plant immunity.

NPR-binding small molecule candidate compounds may be further screenedto determine whether or not NPR-binding is metal dependent, e.g. whetheror not the candidate compound has the ability to coordinate transitionmetals, and in particular, copper. In this regard, binding assays todetermine if the candidate compound binds to a mutated C-terminal NPRactivation region, e.g. including Cys^(521/529) mutations (e.g. mutationof the cysteine or replacement of cysteine with another amino acid toeliminate metal binding), or binds to the C-terminal region of the NPRin the presence of EDTA, may be used. Candidate compounds which do notbind to a mutated C-terminal NPR activation region, or to the C-terminalregion of NPR in the presence of EDTA, exhibit metal dependent NPRbinding and such compounds are candidate compounds that may be useful toactivate PR-1 genes and enhance plant immunity.

Embodiments of the invention are described by reference to the followingspecific example which is not to be construed as limiting.

Example Experimental Procedures Protein Purification for EquilibriumDialysis, ICP-MS and Scintillation Proximity Assay

Proteins were expressed in E. coli as N-terminal fusions to theStrep-Tag according to standard protocols. Recombinant proteins werepurified using 1 ml Strep-Tactin Superflow Plus columns (Qiagen)according to the manufacturer's protocol. The Strep-Tactin buffercontained 50 mM sodium phosphate at pH 8.0 and 300 mM NaCl. For ICP-MSanalyses, the buffer did not contain NaCl and used metal-free water. Forequilibrium dialysis that contained EDTA, bound proteins were treatedwith 10 ml of 50 mM EDTA followed by 10 ml of 5 nM EDTA, prior toelution with a buffer containing 5 nM EDTA. Protein concentrations weremeasured by Bradford assays according to the manufacturer's instruction(Bio-Rad) using BSA as a standard. For metal determination from proteinsexpressed in Arabidopsis, extracts from SA-treated plants wereimmunoprecipitated with an anti-NPR1 antibody as described previously(Rochon et al. 2006. Plant Cell 18, 3670-3685). Protein concentrationswere based on sulfur content determined by ICP-MS. NPR1 was cloned inpGEX-4T-1 as a BamH1/Not1 fragment and expressed as described above.NPR1-GST was purified using 1 ml GSTrap FF column (GE Health) andcleaved on-column using thrombin as described by the manufacturer (GEHealth). The eluted NPR1 was purified by 5300 gel chromatography andrecovered from the void fraction.

Metal-Affinity Chromatography

Proteins were expressed in E. coli as N-terminal fusions to the HA-Tagaccording to standard protocols. Crude lysates were loaded on 1 mlHisTrap FF columns (GE Health) according to the manufacturer's protocol.The HisTrap buffer contained 50 mM HEPES at pH 7.5, 40 mM imidazole, and150 mM NaCl. Where indicated, the HisTrap matrix was stripped of metalusing 10 column-volume of 50 mM EDTA followed by 10 column-volume of 5nM EDTA. Elution was performed in the HisTrap buffer supplemented with 1M imidazole.

Pull-Down Assays

The BTB/POZ (amino acids 1-190 of NPR1) was expressed in E. coli as aC-terminal fusion to the Strep-Tag according to standard protocols. TheΔ513 of NPR1 was expressed as an N-terminal fusion to the HA-Tag asdescribed above. The VLRSgt (as described in Hall et al. (2007) Plant J49, 579-591) was expressed in E. coli as an N-terminal fusion to theGST-Tag according to standard protocols. The pull-down assay wasperformed in the Strep-Tactin buffer. The antibodies used for detectingthe tags in the BTB/POZ-Strep was from Qiagen (catalog #34850) and thoseused for the tags in HA-Δ513 or the GST-VLRSgt were from Santa CruzBiotechnology (catalog #: sc-7392 and sc-138).

Plant Transcription and Two-Hybrid Assays

Arabidopsis thaliana (Columbia) leaves were harvested from four-week-oldplants grown at 21° C. (day) and 18° C. (night) with a ten-hourphotoperiod and transferred to Petri dishes containing MS salts andmicronutrients supplemented with B5 vitamins, 1% sucrose and 0.8% agarat a pH of 5.8. When required, filter-sterilized salicylic acid wasadded to the medium at a final concentration of 1 mM. Coating of thegold particles and general procedures and preparation of the biolisticexperiments were as per the manufacturer's instructions (Bio-Rad). Afterbombardment, leaves were kept in the conditions described above for aperiod of 24 hours before assaying. Enzyme assays were performed usingthe Dual-Luciferase Reporter Assay® system (Promega) following themanufacturer's instructions. Luminescence was measured on a BertholdLumat LB9507 Luminometer (Bad Wildbad, Germany) and the data obtainedrepresented the value of the reporter gene divided by the value of theinternal standard and expressed as Relative Luciferase Units. Toincrease signal-to-noise ratio in some experiments, qPCR was performedto measure the amount of Firefly and Renilla Luciferases mRNA. The datawas reported as Relative Expression and represented the value of thereporter mRNA divided by the value of the internal standard mRNA. Theratio obtained for Gal4 DB was assigned an arbitrary value of 1. One μgof each effector plasmid, 1 μg of the firefly luciferase reporterplasmid, and 0.1 μg of the renilla internal standard plasmid were mixedtogether and the mixture was used to coat beads. This amount of DNA wasused to perform 5 bombardments. Every bar in each graph represents fivebombardments repeated five times on different days (n=25). Theconstructs used contained a Gal4 DB or VP16 N-terminal fusion or nofusion at all.

Equilibrium Dialysis and Scintillation Proximity Assays (SPA)

For equilibrium dialysis (as described in Freifelder, D. (1982).Physical Biochemistry: Applications to Biochemistry and MolecularBiology. W.H.Freeman and Company), two 500 μl chambers (A and B) wereseparated by a dialysis membrane with a cut-off of 3.5 kD. The bufferused in the system was the Strep-Tactin buffer. Radiolabeled SA(PerkinElmer, 50 mCi/mmol) was added in chamber A to a concentration of10 μM SA, calculated based on the total volume of the system (A+B). FourμM of Δ513 protein or 0.8 μM of NPR1 protein were added to chamber B.The system was allowed to equilibrate at 4° C. for 24 hrs. Whereindicated, EDTA was added to both chambers to a final concentration of 5nM. After the 24-hr period, 100 μl from each chamber was removed andcounted for ¹⁴Carbon, allowing for the calculation of SA concentrationin each chamber. Given the dissociation reaction:

Protein-SA_(compiex)

Protein_(free)+SA_(free); the dissociation constant K_(d) equates:

[Protein_(free)]×[SA_(free)]/[Protein−SA_(complex)]. The differentspecies were computed as follow:

[SA_(free)]=[SA_(chamberA)];[Protein−SA_(complex)]=[SA_(chamberB)]−[SA_(chamberA)];

[Protein_(free)]=[Protein_(initial)]−[Protein−SA_(complex)].

For SPA (FIG. 7D-G), radiolabeled SA and NPR1 were incubated with 2 mgof Streptavidin SPA beads (PerkinElmer) in the Strep-Tactin buffer for24 hrs at 4° C. on a rotation wheel. Specific binding was calculated bysubtracting total cpm from non-specific cpm, which were counted byadding a 10-fold excess of cold SA.

For the saturation binding curves (FIGS. 1C and 7D/E), 0.8 μM of NPR1protein was incubated with a final concentration of 0.007-14 μM [¹⁴C]SA.The data was analyzed by non-linear regression using GraphPad PRISM 4and fitted to a one-site-binding rectangular hyperbola. For homologousand heterologous competitive binding curves (FIGS. 1E/F and 7F/G), 0.08μM of NPR1 protein was incubated with a final concentration of 0.07 μM[¹⁴C]SA. Competitors were used at 0.1-100 times the concentration of hotligand, except for BTH, INA, 5-CSA, 4-CSA, and 3,5-DCSA, which were usedat 0.1-10 times the concentration of hot ligand, due to their lowsolubility in water.

Inductively Coupled Plasma-Mass Spectrometry (ICP-MS)

Optimized ICP-MS determinations were performed in a fashion previouslydescribed (Wang et al. (2011) J. Anal. At. Spectrom). Sulfurdeterminations were made using the Dynamic Reaction Cell ICP-MS withchemical resolution, facilitated by using oxygen to generate SO⁺. ICP-MSintensities were converted to concentrations using calibration curves(Table S2). Protein concentrations were based on sulfur content.Proteins were hydrolysed in 68-70% nitric acid for 40 min and thendiluted 40 times in metal-free water before analysis. The StrepTactinbuffer run through the FPLC and through an empty (protein-free)StrepTactin column served as a baseline for metal contamination. ICP-MSintensities of the baseline were subtracted from those of the proteinextracts.

Chromatography

Strep-tagged purified proteins in a final volume of 2 ml were subjectedto gel filtration analysis on the Sephacryl S100 HR or Sephacryl S300 HRpacked in 50 cm long HR 16 columns (GE Health) and equilibrated withS300 chromatography buffer (50 mM HEPES, pH 7.4, 250 mM NaCl. Elutions,in 0.5 ml fractions, were performed in the same buffer at a flow rate of0.8 ml/min. Where indicated, proteins were incubated with 1 mM SA, 1 mMcatechol, 1 mM 4-hydroxy benzoic acid, or 1 mM methyl-salicylate at roomtemperature for 30 min prior to chromatography as described above withthe exception that the chromatography buffer was supplemented with 1 mMSA, 1 mM catechol, 1 mM 4-hydroxy benzoic acid, or 1 mMmethyl-salicylate, respectively. In the case of the EDTA treatment, NPR1was stripped of its metal by a 50 mM EDTA treatment of 30 min, followedby an incubation of 30 min with 1 mM SA prior to gel filtration. In thiscase, the chromatography buffer was supplemented with 1 mM SA.

Quantitative Reverse-Transcriptase Polymerase Chain Reaction

Total RNA was extracted from leaves using the Rneasy plant mini kit(Qiagen) according to the supplier's instructions. After treatment withDnase I (Invitrogen), first strand cDNA synthesis was generated usingSuperScript II reverse transcriptase (Invitrogen), and the (dT)₁₇VNoligo in the presence of 0.4 U Rnasin (Fisher Scientific). Thenewly-synthesized cDNA was diluted 1/200 to reflect a concentration of10 ng μL⁻¹ input total RNA. RT-PCR was performed on a CFX96spectrofluorometric thermal cycler (BioRad). Firefly luciferase valueswere normalized against Renilla Luciferase and PR-1 values againstUbiquitin5 (see Table 1 for primer sequence). All chemicals were used ata concentration of 300 μM, except for BTH, which was used at 100 μM dueto its lower solubility in water. All treatments were for 12 hrs. Valuesconsist of n=3 biological replicates and represent averages±1 SD.

Cross-Linked-Chromatin Chromatography (3C Method)

Plant treatment, cross-linking, sonication, and cross-linking reversalwere performed as for chromatin-immunoprecipitation (Rochon et al.,2006). Chromatography was as described under “Chromatography”. qPCR wasperformed with PR1 and Ubiquitin5 primers. PR-1 values were normalizedagainst against Ubiquitin5 (see Table 1 for primer sequence).

TABLE 1  PCR Primers Used in this Study. *Sequence in 5′ to 3′ DirectionPrimers used for qRT-PCR PR1F GCTCTTGTAGGTGCTCTTGTTCTTCC (SEQ ID NO: 1)PR1R AGTCTGCAGTTGCCTCTTAGTTGTTC (SEQ ID NO: 2) UBQ5-1ACCTACGTTTACCAGAAAGAAGGAGTTGAA (SEQ ID NO: 3) UBQ5-2AGCTTACAAAATTCCCAAATAGAAATGCAG (SEQ ID NO: 4) Primers used for 3C MethodPR1a(−734) GATCACCGATTGACATTGTA (SEQ ID NO: 5) PR1b(−833)GAACACAAAAGTAGATCGGT (SEQ ID NO: 6) UBQ5aGACGCTTCATCTCGTCC (SEQ ID NO: 7) UBQ5bGTAAACGTAGGTGAGTCCA (SEQ ID NO: 8) Primers used for Luciferase qRT-PCRFLucF AGGTGGCTCCCGCTGAATTG (SEQ ID NO: 10) FLucRCATCGTCTTTCCGTGCTCCA (SEQ ID NO: 11) RLucFGTGGTAAACCTGACGTTGTA (SEQ ID NO: 12) RlucRCTTGGCACCTTCAACAATAG (SEQ ID NO: 13) *All PCR primers were synthesizedby Integrated DNA Technologies, Inc.

Data Analysis

All graph results relating to Relative Luciferase Units and mRNARelative Expression are reported as mean±1 standard deviation (SD) of 25independent experiments. Comparisons were performed using two-tailedpaired Student's t test. *p<0.05.

Results NPR1 Binds Specifically to SA

To test whether NPR1 can bind SA directly, a classic method to assessK_(d) values was used, which involved coupling NPR1 to a solid phase andincubating it with radiolabeled SA, followed by washes to remove unboundligand and counting the amount of ligand bound to NPR1. This method didnot yield a measurable apparent equilibrium dissociation constant(K_(d)) as the binding of SA to NPR1 was not above that observed with asolid phase containing no protein (FIG. 1A).

In light of the possibility that the washes alone might be sufficient tore-equilibrate SA between the solid and mobile phases, an equilibriummethod that would avoid such a potential shortcoming was used. Usingequilibrium dialysis (Freifelder, (1982), ibid; Piscitelli et al. (2010)Nature 468, 1129-1132), it was determined that NPR1 and radiolabeled SAcould interact with each other, the amount of SA bound to NPR1 beingclose to 4-orders of magnitude above a no-protein experiment (FIG. 1B).From these data, a low apparent K_(d) of 140±10 nM could be calculated(137±13 nM using the saturation curve in FIG. 1C). The data wasbest-fitted to a single-site-binding rectangular hyperbola (FIG. 1C),indicating that SA binds to one class of binding sites in NPR1. Themaximum binding (B_(max)) was 0.96±0.01 mol SA per mol NPR1.

It was also tested which of the two domains (BTB/POZ or C-terminaltransactivation domain (construct Δ513)) can directly interact with SA.The data demonstrated that the binding affinity of Δ513 (K_(d) of1.49±0.02 μM) for SA is more than 2-orders of magnitude above that ofthe BTB/POZ (K_(d) of 597±14 μM) (FIG. 1B). The NPR1 K_(d) is comparableto the K_(d) found for other plant-hormone receptor-ligand interactionsand is in accordance with the in vivo SA concentration of 0.36 μM (0.05μg/g FW) reported in unstimulated Arabidopsis cells and 7.24 μM (1 μg/gFW) after challenge with an avirulent strain of Pseudomonas syringae.

Chemical specificity of SA-binding was also demonstrated with homologousand heterologous competitive binding curves (FIGS. 1E and F), whichindicated that the structurally-related inactive analogs (FIG. 1G),i.e., catechol, methyl-salicylate, 4-hydroxy benzoic acid, and 3-hydroxybenzoic acid, do not interact with NPR1 with the same affinity as SA. Incontrast, the structurally-related active analogs of SA (FIG. 1G),4-chloro SA, 5-chloro SA and 3,5-dichloro SA, could bind NPR1 with asimilar or slightly better affinity than SA, consistent with theircapacity to trigger PR-1 expression in Arabidopsis (FIG. 1H). Thisexcellent affinity, saturability, and chemical specificity of NPR1 forSA support a model in which NPR1 is an SA-receptor.

From these data, one can deduce that an electronegative functionalgroups, such as a hydroxyl group, in ortho position to anotherelectronegative functional group, such as a free carboxylate, on thearomatic ring are two structural elements required for binding to NPR1.With this in mind, one can predict that the synthetic SAR and PR-1expression inducer benzo(1,2,3)thiadiazole-7-carbothioic acid S-methylester (BTH) would bind NPR1, since it contains two sulfur atoms inpositions geometrically equivalent to the oxygens in the carboxylate andhydroxyl group of SA (arrows on BTH; FIG. 1G). Indeed, BTH does bindNPR1 with a similar or slightly better affinity than SA (FIG. 1E),consistent with its capacity to induce PR-1 expression in Arabidopsis(FIG. 1H). However, a look at 2,6-dichloroisonicotinic acid (INA)reveals that it is similar to 3,5-dichloro SA, but that it is lackingthe hydroxyl group (arrow on INA; FIG. 1G). Therefore, INA would not bepredicted to bind NPR1 and indeed it did not (FIG. 1E). qRT-PCR revealsthat INA was a poor inducer of PR-1 expression in Arabidopsis (FIG. 1H),42 times less effective than an identical concentration of SA and 10times less effective than an identical concentration of the weakestactive SA analog, 4-chloro SA (FIG. 1I). These data suggest that INA mayactivate PR-1 through a mechanism different from that of SA. The bindingdata in FIG. 1 have been validated by a second approach, scintillationproximity assay (FIG. 7).

NPR1 Binds SA Through Cys^(521/529) Via the Transition Metal Copper

It has been demonstrated that Cys^(521/529) of NPR1 are required, alongwith SA treatment, for the activation of PR-1 in vivo and for thetransactivating function of Δ513 and the full-length NPR1. Since SA cancoordinate transition metals through its oxygen atoms, it was determinedwhether or not Δ513 could interact with a transition metal and whetherthis interaction would be dependent on Cys^(521/529). To do so, Δ513fused to an HA-tag was passed through an immobilized metal-affinitycolumn bound to Ni²⁺ (Ni-NTA) and eluted with imidazole. Despite theabsence of a 6-histidine-tag on Δ513, the protein interacted with themetal-bound matrix and was eluted with imidazole just like a His-taggedprotein would (FIG. 2B). Chelation of the Ni²⁺ by EDTA concomitant withthe extraction of the HA-tagged Δ513 abolished the recruitment to theNTA matrix (FIG. 2C), demonstrating that the binding of this protein ismetal-dependent. The recruitment of this protein to the Ni-NTA matrixwas also abolished when Cys^(521/529) were both mutated to non-metalbinding amino acids, e.g. serine residues (Δ513 C521S/C529S) or if theprotein was further deleted by 20 amino acids (Δ533), suggesting thatCys^(521/529) are critical to the transition-metal-binding activity ofNPR1 (FIGS. 2D and E).

To further confirm that SA is perceived through cysteines in ametal-dependent manner, the capacity of both full-length NPR1 and Δ513harboring C-to-S mutations at Cys^(521/529) to interact with SA, usingequilibrium dialysis was tested. In addition, the binding of SA towild-type Δ513 was also evaluated in the presence of EDTA (FIG. 2F).Both metal chelation and the Cys^(521/529) mutations drastically reducedthe SA binding to the C-terminus of NPR1 by several orders of magnitude(FIG. 2F). Using these data, an apparent K_(d) of 1.23±0.3 mM for Δ513 Cto S, and ≧125 mM for Δ513+EDTA, could be calculated. These resultssupport a model in which SA binds to NPR1 via Cys^(521/529) through thecoordination of SA by a transition metal.

It was then determined which of the transition metals (defined asd-block elements of the periodic table) that are most commonly found inliving organisms might be associated with NPR1 in vivo. First, Δ513fused to the Strep-tag was extracted from E. coli and purified on aStrepTactin column prior to metal analysis by Inductively CoupledPlasma-Mass Spectrometry (ICP-MS) as shown in Table 2.

TABLE 2 Slopes and coefficients of determination governing the ICP-MScalibration curves of the various elements studied in FIG. 2g and 2h.Experiment 1 Experiment 2 Experiment 1 Experiment 2 FIG. 2g FIG. 2g FIG.2h FIG. 2h Slope Slope Slope Slope Element (cps/nM)⁴ R² value (cps/nM)R² value (cps/nM) R² value (cps/nM) R² value Mn¹ 1879.7 0.99998 1604.10.99997 1600.5 0.99994 1559.6 0.99991 Fe² 490.42 0.99998 122.92 1 121.60.99981 98.839 0.99963 Co¹ 1845.7 0.99999 1553.9 1 1667 0.99961 1548.90.99976 Ni¹ 414.63 0.99869 347.19 0.99888 371.83 0.99984 342.18 0.99978⁶³Cu¹ 1018.3 0.99999 834.14 0.99997 909.93 0.99974 832.01 0.99966 Zn¹276.75 0.99999 236.35 0.99996 263.59 0.99982 238.98 0.99993 S³ 26.6680.99993 26.944 0.99996 1600.5 0.99994 1559.6 0.99991 ¹Elements weredetected under standard mode. ²Fe was detected under DRC mode with NH₃.³Sulfur was used to determine the protein concentration of wild-typeΔ513 and Δ513 bearing cysteine-to-serine mutations at positions 521 and529. Sulfur was detected under DRC mode with O₂. ⁴cps (counts persecond). The equation was calculated by Linear Thru Zero.

The data indicated that the C-terminus of NPR1 associated preferentiallywith the transition metal, copper (FIG. 2G), and that the mutations ofCys^(521/529) severely curtailed the capacity of Δ513 to interact withcopper. Second, full-length wild-type NPR1 was immunoprecipitated fromArabidopsis before metal analysis by ICP-MS. As a negative control,plants expressing a variant of full-length NPR1 lacking Cys^(521/529)was used. The results (FIG. 2H) were consistent with the observationsmade from E. coli-produced proteins in that NPR1 associatedpreferentially with copper and to a lesser extent with nickel. Mutationsof Cys^(521/529) severely curtailed the capacity of NPR1 to interactwith these metals. Contamination by manganese and zinc was present inArabidopsis extracts. However, detection of Mn and Zn did not depend onCys^(521/529).

The Conformation of NPR1 and Δ513 is Altered by SA

To explore the effect of SA on the conformation of NPR1, gel filtrationexperiments were performed (FIG. 3). In the absence of SA, NPR1 elutedin the void volume of a Sephacryl S300 column (FIG. 3A). However, upontreatment with SA, NPR1 redistributed to the included volume (FIG. 3B)with a stoichiometry consistent with that of a dimer (Tables 3 and 4).

TABLE 3 Predicted and Observed Elution Volumes Establishing theStoichiometry of NPR1 in 1 mM SA on the S300 column. Antici- PredictedPredicted pated MW Kav Ve Fraction Species (kDa) LogMW (Predicted) (mL)number NPR1 66 1.819543936 0.377677209 61.60482554 103-4 monomer NPR1132 2.120573931 0.3003125 56.76953123 94 dimer NPR1 198 2.296665190.255057046 53.94106538 88 trimer NPR1 264 2.421603927 0.22294779151.93423692 84 tetramer Comments: In FIG. 3b (NPR1 + SA panel), thehighest amount of NPR1 found in the included volume was in fractions 90and 95. Since the predicted fraction number for the NPR1 dimer is 94 (anumber between 90 and 95), it would suggest that NPR1 exists as a dimerafter SA treatment.

TABLE 4 Elution Fractions and Corresponding Volumes for Gel filtrationAnalyses. Elution volume (ml) Fraction # From To 50 34.567 35.067 5537.117 37.617 60 39.601 40.101 65 42.118 42.618 70 44.635 45.135 7547.119 47.619 80 49.636 50.136 85 52.16 52.66 90 54.644 55.144 95 57.12757.627 100 59.618 60.118 105 62.135 62.635 110 64.659 65.159 115 67.14367.643 Comments: Each fraction contains approximately 0.5 ml. Since theamount of NPR1 was too low to show observable peaks on the chromatogram,this table is provided to facilitate the conversion between fraction #and elution volume. The fraction # corresponds to the fraction # in FIG.3.

Mutations of Cys^(521/529) or chelation of the metal by EDTA abolishedthe NPR1 conformation change observed after treatment with SA (FIGS. 3C,D and E), confirming the requirement for Cys^(521/529) and a metal forSA interaction (FIG. 2F). A chemical specificity test using catechol,4-hydroxy benzoic acid, and methyl-salicylate indicated that theseinactive structural analogs do not alter the conformation of NPR1 (FIGS.3F, G and H), consistent with their reduced capacity to interact withNPR1 (FIG. 1E). Finally, treatment of NPR1 with the reducing agent DTTdid not induce a redistribution of the protein to the included volume(FIG. 31), indicating that reducing conditions are not required orsufficient for the SA-induced NPR1-redistribution observed here. Atypical Coomassie stained gel of the void fraction reveals that NPR1 andNPR1 C521S/C529S were the major protein component of the void (FIG. 3J)and that therefore the oligomers are unlikely to be due to the presenceof contaminating E. coli proteins.

Although there are no decisive methods to test the stoichiometry of aprotein in vivo, it was determined whether or not NPR1-dependentoligomers form on DNA in vivo by combining chromatin cross-linking, gelfiltration, and qPCR (the 3C Method). The rationale was that, if anNPR1-dependent oligomer forms on the PR1 promoter in vivo, the presenceof PR1 should be detectable by qPCR in the void fraction of an S300after the chromatin had been cross-linked and sheared by sonication.FIG. 3K indicates that in wild-type plants (WT), such an oligomer formson the PR-1 promoter (region −734 to −833 of the promoter) in theabsence of SA (water), but not after a treatment with SA. Repeating theexperiment in the npr1-3 mutant background demonstrated that thisoligomer depended on the presence of NPR1. Treatment of wild-typeArabidopsis with the inactive SA analog, 4-hydroxy benzoic acid (4-OHBA), did not reduce the amount of NPR1-dependent oligomer. These in vivodata are consistent with the in vitro data of FIGS. 3A, B and G.Although BTH treatment could not be used in the in vitro chromatographydue to its low solubility in water, it is assumed that BTH would alsodisassemble an NPR1 oligomer since it is an active functional analog ofSA and it can directly bind NPR1 (FIGS. 1E and H). Performing the 3Cmethod on plants treated with BTH revealed that, like SA, this activeanalog also reduced the amount of NPR1-dependent oligomer on the PR-1promoter (FIG. 3K). By contrast, INA, which did not interact with NPR1in vitro and did not activate PR-1 to the same extent as SA or BTH(FIGS. 1E and H), did not affect the NPR1-dependent oligomer on the PR-1promoter (FIG. 3K). This result further suggests that INA may not be afunctional analog of SA.

The conformation of Δ513 was also investigated by gel filtration. Beforeand after SA-treatment, Δ513 was found in the included volume of aSephacryl S100 column (FIG. 4). The stoichiometry of the untreated Δ513was consistent with that of both a dimer and a trimer (FIGS. 4A and B),while the stoichiometry of the SA-dependent redistributed form of Δ513was consistent with that of a dimer (FIGS. 4C/D and Table 5).

TABLE 5 Predicted and Observed Elution Volumes Establishing theStoichiometry of Δ513 in 1 mM SA on the S100 column. Anticipated MW KavPredicted Ve Observed Ve (mL) Species (kDa) LogMW (Predicted) (mL) −SA+SA Δ513 monomer 10.79 1.033021 0.489456 66.17234571 — — Δ513 dimer21.58 1.334051 0.348664 57.25741343 58.78 57.18 Δ513 trimer 32.371.510143 0.266306 52.04251235 52.51 — Δ513 tetramer 43.16 1.6350810.207872 48.34248115 — — Δ513 11× 118.69 2.074414 0.002397 35.3317471135.79 35.79

However, the elution volumes of the dimer in the untreated (58.78 mL)versus the SA-treated (57.18 mL) Δ513 were different and thereforeindicated that these dimer may not have the same conformation. Theelution volume of the SA-dependent dimer was closer to that of thetheoretical dimer (57.26 mL).

SA Disrupts the BTB/POZ-Transactivation Domain Interaction

When tethered to the Gal4 DNA-binding domain (DB) in an in vivo planttranscription assay, the transactivation domain of NPR1 (construct Δ513)can activate transcription in the absence of SA-treatment, but tetheringof the full-length NPR1 did not (FIG. 5A), suggesting the presence of anauto-inhibitory domain in NPR1. Since BTB/POZ domains can beautoinhibitory, it was determined whether or not the NPR1 BTB/POZ caninteract with the NPR1 transactivation domain. A plant two-hybrid systemin the native organism, Arabidopsis, was used where the BTB/POZ wasfused to the DB (POZ:DB) and the Δ513 was fused to the VP16transactivation domain (Δ513:TA) (FIG. 5B). Here the reporter gene wasmonitored through its mRNA as opposed to its enzyme activity, whichprovided a greater signal-to-noise ratio (see Experimental Procedures).BTB/POZ self-association (POZ:DB+POZ:TA) in the absence or presence ofSA served as a positive control. The interaction between the NPR1BTB/POZ and its transactivation domain (POZ:DB+Δ513:TA) was observablein the absence of SA (significantly different from Gal4 DB, p<0.05), butnot after SA-treatment (not significantly different from Gal4 DB,p>0.05), indicating that SA disrupts the BTB/POZ-Δ513 association (FIG.5B).

Given that the plant two-hybrid system is an in vivo method of analysis,an indirect effect of SA on the interaction of the BTB/POZ and theC-terminus of NPR1 cannot be ruled out. Thus, the interaction in vitroin a pull-down assay (FIG. 8) was tested. Because the BTB/POZ was elutedfrom the solid support with the competing ligand, desthiobiotin, but notwith 1 mM SA (FIG. 5C, left panel), it was concluded that SA, at theconcentration tested, did not disrupt the Strep-tag/StrepTactininteraction. The pull-down indicated that the BTB/POZ interacted withΔ513, but that the interaction was disrupted by 1 mM SA (FIG. 5C, rightpanel). No Δ513 could be further eluted by desthiobiotin, indicatingthat SA displaced all of the Δ513 from the solid phase (FIG. 5C, rightpanel). As negative controls, first an unrelated protein (VLRSgt) wasshown not to interact with BTB/POZ (FIG. 5D) and secondly Δ513 was shownnot to interact with the solid support in the absence of BTB/POZ (FIG.5E). Together these data demonstrate that SA directly disrupts theBTB/POZ-Δ513 interaction, which is consistent with the conformationchange of NPR1 and Δ513 brought about by SA (FIGS. 3A and B, FIG. 4).

The NPR1 BTB/POZ Inhibits the Transactivation Potential of Δ513

It was then determined whether or not the BTB/POZ could modulate thetranscriptional properties of Δ513 (FIG. 5F). When Δ513:DB wasco-expressed in Arabidopsis leaves with the BTB/POZ (not fused to anyforeign transcription activation or DNA-binding domain), expression ofthe reporter gene in untreated cells was reduced to background levels.However, the transcription activity of Δ513 in SA-treated cells wasunaffected by the BTB/POZ, consistent with the fact that these twoproteins could only interact in the absence of SA (FIG. 5B). In an invivo plant repression assay, where the reporter gene is first activatedby LexA:VP16 before testing for repression using a Gal 4:DB fusion, theNPR1 BTB/POZ did not appear to repress the promoter back to basal (Gal4DB) level (FIG. 5G). These data revealed the autoinhibitory capacity ofthe BTB/POZ despite it not being an autonomous transcriptionalrepression domain. Therefore, in the absence of SA, the BTB/POZ musthave masked the interface on the C-terminal transactivation domainrequired for its function.

Discussion

Given the saturability by SA, the low K_(d), and the chemicalspecificity of the SA-NPR1 interaction, which are hallmarks of areceptor, NPR1 is undeniably an SA-receptor.

Direct binding of SA by the receptor, NPR1, reorganizes the conformationof an NPR1-dependent oligomer at the PR-1 promoter and abolishes theinteraction between the auto-inhibitory N-terminal BTB/POZ domain andthe C-terminal transactivation domain of NPR1 (FIG. 6). Thus, a clearmechanistic path is established between the sensing of SA by NPR1 andthe unveiling of the NPR1 transcriptional activation domain, aprerequisite to PR-1 gene activation.

1. A method of enhancing plant immunity by PR-1 gene activationcomprising the step of administering to a plant a small molecule thatbinds to an NPR1 protein, or to a functionally equivalent salicylic acidreceptor protein, and disrupts the interaction between the N-terminalBTB/POZ domain and the C-terminal transactivation domain of the NPR1protein.
 2. The method of claim 1, including the additional step ofadministering a transition metal to the plant or plant growing mediumprior to, following or at the same time as administration of the smallmolecule.
 3. The method of claim 2, wherein the transition metal iscopper.
 4. The method of claim 2, wherein the transition metal isadministered in the form of a salt or a chelate.
 5. The method of claim2, wherein the transition metal is administered to the plant growingmedium in an amount ranging from about 9 to 30 ppm.
 6. The method ofclaim 2, wherein the transition metal is administered directly to theplant in an amount of about 50 to 300 micromolar.
 7. The method of claim1, wherein the NPR-binding small molecule has the following generalformula:

wherein X and Y are each an electronegative functional group thattogether can coordinate a transition metal; and ring A is a hydrophobiccyclic core.
 8. A method of identifying a small molecule candidatecompound that enhances plant immunity comprising the step of screening acandidate small molecule compound for binding to a C-terminaltransactivation domain of an NPR1 protein, or of a functionallyequivalent salicylic acid receptor protein, and determining whether ornot the small molecule binds to the C-terminal transactivation domain,wherein a compound that exhibits a binding affinity for the C-terminaltransactivation domain is a candidate compound that may enhance plantimmunity.
 9. The method of claim 8, additionally comprising the step ofdetermining whether or not binding of the small molecule to theC-terminal transactivation domain is metal dependent, wherein metaldependent binding indicates that the compound is a candidate compoundthat may enhance plant immunity.
 10. The method of claim 9, wherein adetermination of metal-dependent binding of the small molecule to theC-terminal transactivation domain is conducted in the absence of ametal.
 11. The method of claim 9, wherein a determination ofmetal-dependent binding is conducted in the presence of a metalchelator.
 12. The method of claim 9, wherein a determination ofmetal-dependent binding is conducted in the presence of a C-terminaltransactivation domain in which Cys^(521/529) of the transactivationdomain are mutated to non-metal binding amino acids.
 13. The method ofclaim 9, wherein the determination of metal-dependent binding isconducted in the presence of a truncated C-terminal transactivationdomain in which a region including Cys^(521/529) of the transactivationdomain is removed.
 14. The method of claim 8, wherein the compoundexhibits a binding affinity for the C-terminal transactivation domain ofabout 50 μM or less.
 15. The method of claim 9, wherein the compoundexhibits a binding affinity for the C-terminal transactivation domain ofabout 10 μM or less.
 16. The method of claim 8, wherein the compound hasthe general formula:

wherein X and Y are each an electronegative functional group thattogether can coordinate a transition metal; and ring A is a hydrophobiccyclic core.
 17. The method of claim 7, wherein X and Y areindependently selected from the group consisting of hydroxyl, carbonyl,amine, nitro, —SH, —COH, —OCOH, —CH₂OH and —NHR, wherein R is a loweralkyl group.
 18. The method of claim 7, wherein ring A is selected fromthe group consisting of phenyl, heterocyclyl, cyclohexyl andcyclopentyl, optionally substituted with one or more groups selectedfrom halogen, hydroxyl, thio, C₁-C₆ alkyl, C₁-C₆ alkyl halide, —OR¹,—NH₂, —NO₂, —NHR¹, —NR¹R² or —SR¹, fused phenyl and fused heterocyclyl,wherein variables R¹ and R² are independently selected from the groupconsisting of C₁-C₆ alkyl, C₁-C₆ alkyl halide, C₁-C₆ alkanol and C₁-C₆alkoxy.